No effective therapeutic agents currently exist to improve outcomes in severe autism. We recently identified a new cellular mechanism in autism involving endosomal Na+/H+ exchanger 6 (NHE6). Studies of NHE6 offer a valuable opportunity for rapid development of innovative treatment strategies in severe autism and related dis- orders. Human mutations in endosomal NHE6 constitute a newly recognized, monogenic, autism-related disorder. Also, down-regulation of NHE6 gene expression is evident in postmortem brains in approximately 30% of patients with idiopathic autism. NHE6 is an integral membrane protein that regulates proton efflux out of endosomes. We have reported that loss of NHE6 in neurons leads to over-acidification of the endosome lumen, and diminished neuronal arborization and synapse development. We also have exciting, new data demonstrating that intra-endosomal pH in the mutant is normalized in vitro by FDA-approved medicines known to alka- linize endosomes. The objective for this research proposal is to elucidate the mechanisms by which NHE6 mu- tations lead to defects in circuit development, and to test the ability of alkalinizing agents to normalize these mutant phenotypes in mouse in vitro and in vivo. Our data support the following central hypothesis: Loss of NHE6 function leads to over-acidification of early endosomes, which enhances degradation of cargo. We also hypothesize that over-acidification of early endosomes drives trafficking to the degradative pathway (i.e., to late endosomes and lysosomes), and decreases endosome recycling and endosome signaling. Our data indicate that TrkB is cargo in NHE6-associated endosomes. We find attenuated TrkB signaling in NHE6 mutant neu- rons. The BDNF/TrkB pathway is well-known to govern circuit development via endosomal signaling, and has broad significance in neuropsychiatry. Our Aims include: 1) Determine the functional domains of NHE6 protein through study of patient mutations; 2) Determine the role of NHE6 and intra-endosomal pH regulation in TrkB trafficking and endosomal signaling; and 3) Determine the reversibility of defects in neuronal arborization and synapse development in NHE6 mutant neurons using alkalinizing medicines. These studies on the role of intra-endosomal proton concentration are innovative because they represent: 1) a new level of cellular analysis in endosome trafficking and neuronal differentiation; and 2) a novel, druggable cellular mechanism in autism and related disorders. This research is significant because it will lead to 1) elucidation of fundamental and disease- relevant mechanisms in endosome biology and circuit development; and 2) development of specific avenues for mechanism-based treatments for severe autism. This project is also a part of an integrated translational ap- proach in our research group that is coordinated with ongoing studies in patient-derived induced pluripotent stem cells (iPSCs) and with clinical studies in patients with NHE6 mutations. Finally, this application is strongly in line with the NIH Interagency Autism Coordinating Committee (IACC) Strategic Plan that calls for the identification of molecular targets amenable to interventions.